The V antigen is a 37-kDa secreted polypeptide encoded on the 70-kb virulence plasmid of pathogenic Yersinia spp. Besides having regulatory functions, it is known to be a virulence factor and a protective antigen. DNA sequencing of the most common serotypes of human pathogenic Yersinia enterocolitica and Y. pseudotuberculosis revealed that two evolutionary distinct types of V antigen exist in Yersinia spp. One type is represented by Y. enterocolitica serotype 08 strains WA, WA-314, and NCTC 10938 (designated LcrV-YenO8); the other type comprises Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serotypes O3, O9, and O5,27 (LcrV-Yps). A hypervariable region between amino acids 225 and 232 represents the main difference between the two types. By raising monospecific antisera against both types of V antigen (anti-rVO8 and anti-rVO3), we were able to demonstrate that, in general, passive immunization of mice against a challenge with yersiniae was possible with both anti-Y. enterocolitica V antigen sera. However, anti-V antigen serum was protective only if the immunizing V antigen was the same type as the V antigen produced by the infective strain. The failure of the American V antigen type represented by Y. enterocolitica serotype O8 to protect against Yersinia spp. carrying the other V antigen type (LcrV-Yps) could be an explanation for the presence of plague foci in American countries.